Synthetic Nanopores for DNA Sequencing

ABSTRACT

A compound and methods of making thereof having the structure shown below is disclosed. Each Ar is an aromatic group. Each M is palladium, platinum, or rhenium. At least one X in the compound has an aliphatic having at least 1 carbon atom. Each x, each y, and each z is an integer greater than or equal to zero. Each m is an integer greater than or equal to one. n is an integer greater than or equal to three.

This application is a divisional application of U.S. patent applicationSer. No. 11/070,397 filed on Feb. 28, 2005 and allowed, which claimspriority to U.S. Provisional Patent Application No. 60/550,739 filed onMar. 1, 2004 and to U.S. Provisional Patent Application No. 60/559,288filed on Mar. 31, 2004, all incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is related to cyclic oligomeric compounds.

2. Description of Related Art

There are two approaches in nanopore sequencing. In the first approach,the proteinaceous pore, alpha-Hemolysin, is used to reconstitute aplanar phospholipid bilayer membrane. A black lipid membrane (BLM) isformed on a thin Teflon partition with a hole in the middle several tensof microns in diameter. The partition separates identical aqueous media(e.g. 1 M KCl with buffer at neutral pH) in a trough. A voltage isapplied so as to drive an ionic current through the open pore. If singlestranded DNA is introduced into the cis chamber (the chamber with thenegative electrode) current blockades are observed during polyanionicDNA translocation through the protein pore into the opposite transchamber. (Henrickson et al., “Driven DNA Transport into an AsymmetricNanometer-Scale Pore,” Phys. Rev. Lett., 85, 3057 (2000). All referencedpublications and patent documents are incorporated herein by reference.)The limitations with this approach are that 1) proteins are fragile, 2)scaling up for industrial production is not feasible 3) the nanopore ishighly asymmetric in size and interactions 4) conformationalfluctuations within the pore raise the noise level.

To eliminate the problems associated with fragile pores, solid statemembranes (such as silicon nitride) were ion beam milled to form a porefor nanopore sequencing (Li et al., “DNA Molecules and Configurations ina Solid-State Nanopore Microscope,” Nature Mat., 2, 611 (2003)). Thedisadvantages with this approach are 1) it is difficult to routinelyprocess holes less than 40 nm in diameter 2) reproducibility of the porefeatures is difficult 3) the membrane thickness is much larger than thesize of a DNA nucleotide 4) interaction between the translocating DNAand the pore cannot be tuned for controlling the translocation (otherthan through physical variables such as voltage and concentration ofsolution).

SUMMARY OF THE INVENTION

The invention comprises a compound comprising the formula in Eq. (1).Each Ar is an independently selected aromatic group, heteroaromaticgroup, or polyaromatic group. Each M is independently selected from thegroup consisting of palladium, platinum, and rhenium. Each X is anindependently selected chemical group or bidentate chelating ligand,with the proviso that at least one X in the compound comprises analiphatic having at least 1 carbon atoms. Each x, each y, and each z isan independently selected integer greater than or equal to zero. Each mis an independently selected integer greater than or equal to one. n isan integer greater than or equal to three.

The invention further comprises a compound comprising the formula in Eq.(2). Ar, x, y, z, and m are as defined above. Z is an independentlyselected chemical group or bidentate chelating ligand. Each x′, each y′,and each z′ is an independently selected integer greater than or equalto zero.

The invention further comprises a method of making a compound comprisingproviding a spacer compound comprising the formula in Eq (3) andreacting it with a metal compound comprising the formula Y₂MX_(m). Ar,x, y, z, M, X, and m are as defined above. Each Y is an independentlyselected leaving group capable of facilitating the formation of a N-Mbond with a pyridinyl nitrogen.

The invention further comprises a method of making a compound comprisingproviding a 90° angled compound comprising the formula in Eq. (2) andreacting it with a metal compound comprising the formula Y₂MX_(m). X, m,and Y are as defined above. M is palladium or platinum.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention will be readily obtainedby reference to the following Description of the Example Embodiments andthe accompanying drawings.

FIG. 1 schematically illustrates a compound of the invention in a lipidbilayer.

FIG. 2 shows a Langmuir Blodgett isotherm of a pure nanopore compound.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

In the following description, for purposes of explanation and notlimitation, specific details are set forth in order to provide athorough understanding of the present invention. However, it will beapparent to one skilled in the art that the present invention may bepracticed in other embodiments that depart from these specific details.In other instances, detailed descriptions of well-known methods anddevices are omitted so as to not obscure the description of the presentinvention with unnecessary detail.

The approach disclosed can utilize robust, synthetic organic nanoporesto replace the fragile proteinaceous ion pores currently used forstudying DNA translocation. The interaction forces between thetranslocating molecule and the organic nanopore may be tuned(theoretically predicted to control biomolecule translocation) byengineering the pore (size and chemistry) to achieve single base pairresolution.

A nanoscale interface technology is schematically illustrated in FIG. 1.A bilayer lipid membrane 10 is reconstituted with the organic nanopore20. This can form the partition in a nanopore sequencer to replace thedelicate protein pores currently in use. The organic nanopore approachmay afford the capability to fabricate nanopores at the size scalerequired for high resolution sequencing, for example, in the 1 to 2 nmrange. It is currently impossible to routinely machine holes of thatsize in a silicon nitride membrane, for example.

Possible characteristics of the organic nanopores may include: 1)robust, 2) reproducible, 3) one-time synthesis is sufficient since alarge number of pores are available even in a milligram of material, 4)amenable to industrial scale production, 5) provide a symmetric barrier,6) size and chemistry can be varied to tune DNA pore interaction (ratherthan varying physical variables such as voltage and concentration ofsolution), 7) conformational fluctuations within the pore eliminated 8)temperature of the nanopore assembly can be varied over a wide range, 9)capable of metal directed self assembly with high or 100% yield, 10)height of the nanopore is far lower than protein pore (the nm height ofthe protein channel forces several base pairs to be within the channel.This causes the electrical signatures characteristic of the base pairsto be an average over several base pairs. By using a shorter channel asin the organic nanopore approach, such averaging is avoided, permittingelectrical signatures to be more representative of single base pairs.Statistical analysis can therefore be used readily to extract signaturesindicative of the type of translocating nucleotide.)

The compounds of the invention may have a generally polygonal shape,such as a square when n is 4 or triangle when n is 3. The cornerscontain a metal atom having one or more ligands. At least one of theligands in the entire compound is an aliphatic group. Between the metalatoms are spacers that form the edges of the polygon. When spacers are achain of p-phenyl groups and ethyl groups and they are relativelystraight and rigid. The spacers terminate in p-pyridyl groups. Suitablespacers include, but are not limited to, those shown in Eq. (4). Thesespacers may be made by methods known in the art. Generally, the longerthe spacer, the larger the opening in the center of the compound.

The general formula for the spacer is shown in Eq. (3). This generalformula, above and as used in the claims, encompasses additionalsubstituents on the aromatic rings, including the pyridine rings. Insome embodiments, all the Ar groups are phenyl and all the substituentsare hydrogen. The variable z may be the same or different in each spacerin the compound. The variables x and y may be the same or different ineach repeat unit within a spacer. For example, in1,4-bis(4-ethynylpyridine)benzene, z is 2. In one repeat unit x is zeroand y is one. In the other repeat unit, both x and y are one. When allthe spacers are 1,4-bis(4-ethynylpyridine)benzene, the compound is asshown in Eq. (5).

In one embodiment, the spacer is reacted with a metal compound. Themetal compound contains the ligands described above, as well as aleaving group. The leaving group takes part in a reaction where acoordinative bond is formed between the metal and the nitrogen in thespacer and the leaving group is removed from the metal. Such leavinggroups are known in the art. The leaving group may have a negativecharge after leaving. The metal atom may also have a positive charge.The leaving group may then be the anion of a salt. Suitable leavinggroups include, but are not limited to, triflate and carbon monoxide.Other counterions include, but are not limited to, nitrate,tetrafluoroborate, and hexafluorophosphate.

The metal compound may have two of the same leaving group or twodifferent leaving groups. When the leaving group bonds in the metalcompound are at a less than 180° angle to each other, the reaction canproduce a cyclic compound. For example, when the leaving groups are at aright angle, then four metal compounds may react with four spacers ofthe same length to form a square shaped molecule.

The metal compound may also contain other ligands besides the leavinggroups. Any combination of ligands, whether the same or different may beused, as long as the ligands do not prevent formation of themetal-nitrogen bond. At least one of the ligands contains an aliphaticgroup. Suitable ligands include, but are not limited to,trialkylphosphanyl, trioctylphosphanyl, bromide, and carbon monoxide.The ligand can comprise an aliphatic having at least 6 carbon atoms. Theligand may also be a bidentate chelating ligand including, but are notlimited to 1,2-bis(dioctadecylphosphino)ethane.

Suitable metal compounds include, but are not limited to,bis(trioctylphosphino)-platinum(II)bis(trifluoromethanesulphonate) andpentacarbonylrhenium bromide. Another suitable metal compound isbis(trialkylphosphino)-platinum(II)bis(trifluoromethanesulphonate) orPt(PR₃)₂(OSO₂CF₃)₂ where each R group is independently selected. Twoexample compounds are shown in Eq. (6). These compounds may be inequilibrium with each other.

The compound may be made with all identical spacers and identical metalcompounds. Alternatively, combinations of different spacers and metalcompounds may be used within one compound. The different elements may berandomly arranged, or they may form a regular pattern. In oneembodiment, a compound having two different corner groups is made, whereopposite corners have the same group. This may be done in a two-stepreaction. In the first step, a spacer is reacted with a metal compoundin a 2:1 molar ratio. The product contains two spacers bonded to themetal atom, and may form a right angle. This intermediate is thenreacted with another metal compound in an equimolar ratio. In thisreaction, the two right angle compounds form a complete square. Anexample of this type of compound is shown in Eq. (7).

The compound may be soluble in a lipid membrane, including the blacklipid membranes disclosed in U.S. patent application to Shenoy et al.,“Method of Stabilization of Functional Nanoscale Pores for DeviceApplications,” designated as N.C. 96,168, and filed on the same day asthe present application (incorporated herein by reference). Thesolubility may be enhanced by the aliphatic ligands. A long chainaliphatic, such as octyl, may be rapidly soluble in the long aliphaticchains of lipids. The compound may form a pore through the membrane.This structure may be useful for DNA and other polynucleotidesequencing. An electrolyte solution containing the DNA is placed on oneside of the membrane. Electrolyte is also placed on the other side ofthe membrane. A voltage is applied through the electrolytes and acrossthe membrane. This causes a DNA strand to gradually pass through themembrane. As the strand passes through, the current passing through themembrane is measured. The current is affected by the number and identityof the nucleotides presently in the pore. When using protein ionchannels, there is typically more than one nucleotide in the pore. Theidentity of each nucleotide is determined from several currentmeasurements as the nucleotide passes through the pore. A synthetic poremay be short enough to hold only one nucleotide. This simplifies thesequencing, as each nucleotide identification is determined from asingle current measurement.

This approach may enable high selectivity, sensitivity, and real-timemolecular recognition for a variety of target molecules of interest suchas DNA. RNA, proteins, and ions. The proposed method can have thepotential to sequence single stranded DNA (ssDNA) and RNA withresolution at the single base level, with similar capabilities forproteins (at the single amino acid level) and individual ions.

The compounds may enable modification of the interaction forces at themolecular level through molecular engineering of the organic nanopore.This ability translates into a handle on the translocation ofbiomolecules (residence time of base pairs within the pore can becontrolled) with potential for single base pair resolution. Further,information on the effect of interferents such as small molecules, ionsetc may be obtained. Similar resolution may be realized for proteins foramino acid sequencing and for ions such as for Ca channels. Also, sincethe organic nanopores can be robust, it may be possible to investigatethe effects of translocation at higher temperatures. The enhanceddiffusion rates at higher temperatures may allow for higher throughputsequencing to be achieved.

Having described the invention, the following examples are given toillustrate specific applications of the invention. These specificexamples are not intended to limit the scope of the invention describedin this application.

EXAMPLE 1

Synthesis of 1,4-bis(4-ethynylpyridine)benzene—This dipyridine spacercompound has been reported in the literature (Champness et al., “AnImproved Preparation of 4-Ethynylpyridine and its Application to theSynthesis of Linear Bipyridyl Ligands,” Tetrahedron Lett., 40, 5413-6416(1999)) and was prepared according to known literature procedures. 400mg of 4-ethynylpyridine hydrochloride (2.87 mmol), 451 mg of1,4-diiodobenzene (1.37 mmol), 20 mg ofbis(triphenylphosphino)palladium(II)dichloride (0.028 mmol), and 7 mg ofcopper(I)bromide were suspended in 10-15 mL of triethylamine and heatedto 60° C. for 1 hour while stirring vigorously. Then the mixture washeated further and kept at reflux temperature for 48 hours. Subsequentlythe triethylamine was removed by evaporation under reduced pressure. Theresidue was dissolved in dichloromethane (100-200 mL) and the resultingdark solution was washed with 100 mL of a saturated aqueous solution ofdipotassium carbonate. The dichloromethane fraction was then dried overdisodium sulphate, filtered, and evaporated under reduced pressure. Theresulting crude, dark reaction product was purified by columnchromatography (SiO₂, eluent: ethylacetate/hexane/dichloromethane 2/1/3,the product was photoluminescent upon irradiation with UV light). Yield150 mg (40%).

Characterization: ¹H NMR (300 MHz, CDCl₃, 300K), δ (ppm): 8.61 (dbroadened, J=4.4 Hz, 4H; H_(a)), 7.56 (s broad, 4H; H_(g)), 7.10 (mbroadened, J₁=4.4 Hz, 4H; H_(b)).

EXAMPLE 2

Synthesis of 4,4′-bis(ethynyl-4-pyridine)biphenyl—This spacer wassynthesised by the same procedure as applied in Example 1 using4,4′-diiodobiphenyl instead of 1,4-diiodobenzene. Characterization: ¹HNMR (300 MHz, DMSO-d₆, 300K), δ (ppm): 8.62 (d broadened, J=6.0 Hz, 2H;H_(a)), 7.84 (d broadened, J=8.5 Hz, 2H; H_(h)), 7.71 (m broadened,J=8.5 Hz, 2H; H_(b)), 7.53(m, J₁=6.0 Hz, 2H; H_(g)); ¹³C{¹H} NMR (75MHz, CDCl₃, 300K), δ (ppm): 150.6 (s, 4C; C_(n), 141.6 (s small; C_(i)),133.2 (s, 4C; C_(h)), 132.1 (s small; C_(c)), 127.9 (s, 4C; C_(b)),126.3 (s, 4C; C_(g)), 122.4 (s small; C_(t)), 94.4 (s small; C_(d)),88.5 (s small; C_(c)).

EXAMPLE 3

Synthesis ofbis(trioctylphosphino)-platinum(II)bis(trifluoromethanesulphonate)—Thiscompound was obtained by replacing the chloride ions ofbis(trioctylphosphino)platinum-dichloride with trifluoromethylsulphonateions (CF₃SO₃ ⁻, also known as ‘triflate’). 57 mg ofbis(trioctylphosphino)platinum(II)dichloride (0.057 mmol) were dissolvedin 10 mL of dichloromethane. 29.1 mg ofsilver(I)trifluoromethanesulphonate (0.113 mmol) were added and thesolution was stirred for about 1 hour under exclusion of light. Thewhite precipitate (silver(I)chloride) was filtered off and the solutionwas evaporated under reduced pressure. Yield: 68 mg (97%).

EXAMPLE 4

Synthesis of compound in Eq. (6)—1,4-Bis(4-ethynylpyridine)benzene andbis(trioctylphosphino)platinum-bis(trifluoromethylsulphonate) were mixedin equimolar amounts in a chloroform solution. The two forms (square andtriangle) were in a dynamic equilibrium in solution, with asquare:triangle ratio of 3:7. In the square, the distance from onecorner to the opposite corner was 2.2 nm. In the triangle, distance fromone corner to the opposite edge was 1.7 nm. The solid state compositionof the assemblies is unknown.

Characterization: ¹H NMR (300 MHz, CDCl₃, 300K), δ ppm: spacer: square)9.35 (d broad, J=5 Hz, 16H; H_(a)), 7.65 (m, broad, partially belowsignals of triangle, 16H; H_(b)), 7.51 (m, partially below signals oftriangle, 16H; H_(g)); triangle) 9.32 (d, broad, J=5 Hz, 16H; H_(a)),7.60 (d, J=6 Hz, 16H; H_(b)), 7.51 (s, broad, 16H; H_(g)); phosphinealkyl chains for square and triangle: (1.7, m, very broad, ˜96H; CH₂ αand β), 1.2 (s, very broad, ˜240H; CH₂ γ-η), 0.85 (t broad, J≈5 Hz,˜72H; CH₃θ); ³¹P NMR (161.98 MHz, CDCl₃, 300K), δ ppm: −13.84 (¹Jcoupling with ¹⁹⁵Pt (33.8% abundance): 3122). (Subscripts a-n and α-θare as in similar compound in Eq. (7).)

EXAMPLE 5

Synthesis ofbis(1,4-bis(ethynyl-4-pyridine)benzene)rhenium(I)tricarbonylbromide—9.9mg of pentacarbonylrhenium bromide (Re(CO)₅Br, 0.024 mmol) and 15.0 mgof 1,4-bis(4-ethynylpyridine)benzene (0.054 mmol) were heated inisooctane at 60° C. for 36 hours. The formed precipitate was filtered(or decanted) and was washed once with 3-4 mL of isooctane. After dryingunder high vacuum the resulting product was found to be >95% pure by ¹HNMR, Yield: 95%. Characterization: ¹H NMR (300 MHz, CDCl₃, 300K), δ ppm:8.81 (d, J=6.6 Hz, 4H; H_(a)), 8.64 (d, J= 6.0 Hz, 4H; H_(n)), 7.59 (s,broad, 8H; H_(g) and H_(h)), 7.44 (d, J=6.0 Hz, 411; H_(m)), 7.40 (d,J=6.6 Hz, 4H; H_(b)).

EXAMPLE 6

Synthesis of compound in Eq. (7)—The compound was prepared by mixingbis(spacer)rhenium(I)tricarbonylbromide ((spacer)₂Re(CO)₃Br) andbis(trioctylphosphino)-platinum(II)bis(trifluoromethancsulphonate) inequimolar amounts in a chloroform solution. The compound was soluble indimethylsulfoxide (DMSO), and slightly soluble in chloroform or acetone.The distance from one corner to the opposite corner was 2.2 nm.Molecular weight: 4287.5 g/mole.

Characterization: ¹H NMR (300 MHz, CDCl₃, 300K), δ ppm: 9.35 (s, broad,8H; H_(n)), 8.79 (s, broad, 4H; H_(a)), 7.66-7.51 (m, 16H; H_(g) andH_(h)), 7.54 (s, broad, 8H; H_(m)), 7.39 (s, broad, 8H; H_(b)), 1.94 (s,very broad, 24H; PR₃H_(α)), 1.63 (s, very broad, 48H; PR₃H_(β) andH_(γ)), 1.26 (s, very broad, 96H; PR₃H_(δ) to H_(η)), 0.87 (s, broad,36H; PR₃ methyl H_(θ)). (Subscripts a-n and α-θ are as in Eq. (7).)

EXAMPLE 7

Nanopore-lipid compatibility experiments—To demonstrate that thesynthetic nanopore will partition into lipid membranes to form singlechannels, Langmuir Blodgett (LB) isotherms were generated of the purenanopore material as well as mixtures of nanopores and lipids. FIG. 2shows an example of an isotherm from which the cross-sectional area ofthe pore was calculated. The calculated size agrees with the sizecomputed based on molecular structure and is large enough for singlestranded DNA translocation. Further, this LB experiment also proved thatthe pore is oriented with the channel axis parallel to the long axis ofthe phospholipid molecules.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that the claimed invention may be practiced otherwise than asspecifically described.

1. A compound comprising the formula:

wherein each Ar is an independently selected aromatic group,heteroaromatic group, or polyaromatic group; wherein each x, each y,each z, each x′, each y′, and each z′ is an independently selectedinteger greater than or equal to zero; wherein each Z is anindependently selected chemical group or bidentate chelating ligand; andwherein each m is an independently selected integer greater than orequal to one.
 2. The compound of claim 1, wherein each Ar is phenyl. 3.The compound of claim 1, wherein each X is independently selected fromthe group consisting of bromide and carbon monoxide.
 4. The compound ofclaim 1, wherein the 90° angled compound isbis(1,4-bis(ethynyl-4-pyridine)benzene)rhenium(I)tricarbonylbromide. 5.A method of making a compound comprising: providing a 90° angledcompound comprising the formula:

wherein each Ar is an independently selected aromatic group,heteroaromatic group, or polyaromatic group; wherein each x, each y,each z, each x′, each y′, and each z′ is an independently selectedinteger greater than or equal to zero; wherein each Z is anindependently selected chemical group or bidentate chelating ligand;wherein each m is an independently selected integer greater than orequal to one; and reacting the 90° angled compound with a metal compoundcomprising the formula Y₂MX_(m); wherein each M is independentlyselected from the group consisting of palladium and platinum; whereineach X is an independently selected chemical group or bidentatechelating ligand, with the proviso that at least one X in the compoundcomprises an aliphatic having at least 1 carbon atom; wherein each m isan independently selected integer greater than or equal to one; andwherein each Y is an independently selected leaving group capable offacilitating the formation of a N-M bond with a pyridinyl nitrogen. 6.The method of claim 5, wherein the 90° angled compound isbis(1,4-bis(ethynyl-4-pyridine)benzene)rhenium(I)tricarbonylbromide. 7.The method of claim 5, wherein at least one X in the compound comprisesan aliphatic having at least 6 carbon atoms.
 8. The method of claim 5,wherein the metal compound isbis(trioctylphosphino)-platinum(II)bis(trifluoromethanesulphonate).